2,6-dimethylpyridine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test:

The test substance did not show increase in the number of revertant colonies. Hence, the test susbstance is considered to be non-mutagenic.

Chromosomal aberration assay:

The test substance did not show mutation in human lymphocytes. Hence, the test susbstance is considered to be non-mutagenic.

Mammalian cell gene mutation assay:

The test substance did not show mutation in human lymphocytes. Hence, the test susbstance is considered to be non-mutagenic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Remarks:

Read across data

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Justification for type of information:

Data is from secondary literature

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine gene

Species / strain / cell type:

other: TA97, TA98, TA100, TA102, TA 1535 and/or TA 1537

Details on mammalian cell type (if applicable):

Not specified

Metabolic activation:

with and without

Metabolic activation system:

Not specified

Test concentrations with justification for top dose:

up to 5000 µg/plate

Vehicle / solvent:

Not specified

Untreated negative controls:

yes

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

not specified

Details on test system and experimental conditions:

Not specified

Evaluation criteria:

Not specified

Statistics:

Not specified

Species / strain:

other: TA97, TA98, TA100, TA102, TA 1535 and/or TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

other: Non-mutagenic

Conclusions:

The test substance did not show increase in the number of revertant colonies. Hence, the test susbstance is considered to be non-mutagenic.

Executive summary:

in vitro Gene mutation toxicity study was performed to determine the mutagenic nature of the read across substance 2 -Picoline (CAS no.: 109 -06 -8, E.C. no.: 203 -643 -7). The study was performed using Salmonella typhimurium strains TA97, TA98, TA100, TA102, TA1535 and/or TA1537 in the presence and absence of metabolic activation system. The chemical was used at dose levels upto 5000 µg/plate. Positive and negative controls were tested concurrently. The test chemical did not induce gene mutation in Salmonella typhimurium strains in the presence and absence of metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Remarks:

Read across data

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Justification for type of information:

Data is from secondary literature and from read across substance.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

- Name of the test material: 5-ethyl-2-methylpyridine
- Molecular formula: C8H11N
- Molecular weight: 121.182 g/mol
- SMILES: CCc1ccc(C)nc1
- InChI: 1S/C8H11N/c1-3-8-5-4-7(2)9-6-8/h4-6H,3H2,1-2H3
- Substance type: Organic
- Physical state: Liquid

Species / strain / cell type:

other: human lymphocytes

Details on mammalian cell type (if applicable):

Not specified

Metabolic activation:

with and without

Metabolic activation system:

Not specified

Test concentrations with justification for top dose:

100, 200, 300, 400 μg/ml
Mitotic index reduced by 81% at the highest
concentration

Vehicle / solvent:

Not specified

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Details on test system and experimental conditions:

Not specified

Evaluation criteria:

Not specified

Statistics:

Not specified

Species / strain:

other: human lymphocytes

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at highest dose conc.

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

other: Non-mutagenic

Conclusions:

The test substance did not show mutation in human lymphocytes. Hence, the test susbstance is considered to be non-mutagenic.

Executive summary:

In vitro chromosomal aberration study was performed to determine the mutagenic nature of the read across substance 5-Ethyl-2-picoline (CAS no.: 104 -90 -5, E.C. no.: 203 -250 -0). The study was performed using human lymphocytes in the absence of metabolic activation system. The chemical was used at doses 100, 200, 300 and 400 µg/ml. The test chemical did not induce gene mutation in human lymphocyte cells in the absence of metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Remarks:

Read across data

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The study contains experimental data of a read-across analogue

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Principles of method if other than guideline:

The purpose of this study was to assess toxic and genotoxic effects of the test chemical on Chinese Hamster Ovary (CHO) cells by using several different in vitro-based assays, including genotoxicity tests based on the OECD Guideline No. 476 “In Vitro Mammalian Cell Gene Mutation Test”.

GLP compliance:

no

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

Cells deficient in hypoxanthine-guanine phosphoribosyl transferase (HPRT) due to the mutation HPRT+/- to HPRT-/- are resistant to cytotoxic effects of 6-thioguanine (TG). HPRT proficient cells are sensitive to TG (which causes inhibition of cellular metabolism and halts further cell division since HPRT enzyme activity is important for DNA synthesis), so mutant cells can proliferate in the presence of TG, while normal cells, containing hypoxanthine-guanine phosphoribosyl transferase cannot.

This in vitro test is an assay for the detection of forward gene mutations at the in hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on the X chromosomes of hypodiploid, modal No. 20, CHO cells. Gene and chromosome mutations are considered as an initial step in the carcinogenic process.
The hypodiploid CHO cells are exposed to the test item with and without exogenous metabolic activation. Following an expression time the descendants of the treated cell population are monitored for the loss of functional HPRT enzyme.
HPRT catalyses the transformation of the purine analogues 6-thioguanine (TG) rendering them cytotoxic to normal cells. Hence, cells with mutations in the HPRT gene cannot phosphoribosylate the analogue and survive treatment with TG.

Therefore, mutated cells are able to proliferate in the presence of TG whereas the non-mutated cells die. However, the mutant phenotype requires a certain period of time before it is completely expressed. The phenotypic expression is achieved by allowing exponential growth of the cells for 7 days.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Cell line used: Chinese Hamster Ovary (CHO) cells
- Type and identity of media: Ham's F-12K (Kaighn's) Medium containing 2 mM L-Glutamine supplemented with 10% Fetal Bovine Serum and 1% Penicillin-Streptomycin (10,000 U/mL).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Not applicable
- Periodically checked for karyotype stability: Not applicable

Additional strain / cell type characteristics:

other: Hypodiploid, modal No. 20

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction obtained from Arcolor 1254-induced male Sprague-Dawley rats

Test concentrations with justification for top dose:

0, 0.1, 0.25, 0.5 or 1 mM

Vehicle / solvent:

Vehicle(s)/solvent(s) used: Ethanol
Justification for choice of solvent/ vehicle: Methyl 2-napthyl ether was easily dissolved in ethanol.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

N-ethyl-N-nitrosourea (ENU) was the positive control substance in the tests done without S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium with pre-incubation

DURATION
- Preincubation period: One week involving 3 days of incubation with Hypoxanthine-aminopterin-thymidine (HAT) in medium as a mutant cleansing stage, followed by overnight incubation with hypoxanthine-thymidine (HT) in medium prior to a 3-4 days incubation in regular cell medium. After seeding and prior to treatment, the mutant-free cells were incubated for an additional of 24 hours
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 14 days
- Fixation time (start of exposure up to fixation or harvest of cells): 7 days (harvest of cells)

SELECTION AGENT (mutation assays): 6-thioguanine (TG)
SPINDLE INHIBITOR (cytogenetic assays): Not applicable
STAIN (for cytogenetic assays): Crystal violet

NUMBER OF REPLICATIONS: A minimum of 2 replicates per dose concentration including negative and positive control.

NUMBER OF CELLS EVALUATED: 5 x 10 E5 cells were plated 7 days after treatment and whatever cells left, after 14 days of incubation with the selection medium, were evaluated.

DETERMINATION OF CYTOTOXICITY
- Cytotoxicity test
After being exposed to the test chemical for 3 hours, in the absence or presence of S9, cells were trypsinized and 0.5 x 10 E5 cells per well was seeded in duplicates from two parallel duplicate cultures into 6-well plates in fresh medium. The relative total growth and cytotoxicity was evaluated 24 and 48 hours after seeding.

OTHER EXAMINATIONS: Not applicable

Rationale for test conditions:

No data

Evaluation criteria:

The plates were scored for total number of colonies

Statistics:

No data

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

not valid

Table 1A.Effect of the test chemical ether exposure on gene toxicity in CHO cells. After being exposed to the test chemical for 3 hrs, cells was washed with sterile PBS and then incubated for 7 days at 37°C, 5% CO₂. After 7 days, cells were re-seeded in new 6-well plates in the absence or presence of 10mM TG as a selection agent and returned to the incubator for 14 days at 37°C, 5% CO₂. On day 15, all 6-well plates were stained with crystal violet and the number of colonies were counted manually. The results are presented as the total number of colonies found in the number of independent wells analyzed (e.g. 0 colonies in 4 wells will give 0/4) (n = 2 samples from 2 independent cultures).

 

	With S9		Without S9
	with TG	without TG	with TG	without TG
Neg. control	0/4	195/4	0/4	180/4
Pos. control	0/4	199/4	27/4	142/4
0.1 mM	2^a/4	229/4	0/4	186/4
0.25 mM	0/4	209/4	0/4	142/4
0.5 mM	0/4	186/4	1/4^b	205/4
1.0 mM	0/4	179/4	0/4	137/4

 

^a)Two very diffuse colonies were found in one single well.

^a)One very diffuse colony was found in one single well.

 

 

Table 1B.Mutation frequency in CHO cells after 3 hrs of exposure to Methyl 2-napthyl ether in the absence or presence of 4% S9 liver microsomal fraction. N/A, no colonies present in the samples selected with TG, i.e. no mutation frequency could be determined.

 

	With S9	Without S9
Neg. control	N/A	N/A
Pos. control	N/A	5.35x10^-4
0.1 mM	N/A^a	N/A
0.25 mM	N/A	N/A
0.5 mM	N/A	N/A^b
1.0 mM	N/A	N/A

 

^a)Since two very diffuse colonies were found in one single well (see Table 1A), the diffuse colonies was not regarded as reliable and true colonies since the cells seemed to be apoptotic.

^b)Since only one diffuse colony were found in one single well (see Table 1A), the diffuse colony was not regarded as a reliable and true colony since the cells seemed to be apoptotic.

Conclusions:

In an in vitro mammalian cell gene mutation study, Chinese Hamster Ovary (CHO) cells were exposed to the test chemical in the concentration of 0, 0.01, 0.25, 0.5 or 1 mM in the presence and absence of S9-induced metabolic activation. The results showed no evidence of gene toxicity when cells were exposed to the given test chemical. Therefore, it is not mutagenic.

Executive summary:

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of the given read across analogue 2 -methoxynapthalene (CAS no. 93 -04 -9) when administered to Chinese Hamster Ovary (CHO) cells. The test substance was administered to CHO cells for 3 hours at the dose levels of 0, 0.1, 0.25, 0.5 or 1.0 mM in the absence or presence of exogenous metabolic activation (S9 mix). CHO cells representing the negative controls were exposed to the vehicle. Positive controls, such as N-ethyl-N-nitrosourea (ENU) experiments without metabolic activation and 7,12-dimethylbenz(a) anthracene in experiments with metabolic activation, were also included in each test. The positive control ENU gave a clear indication of gene mutations occurring while no other treatment gave rise to gene toxicity. No mutant colonies were detected following the exposure to positive control of7,12-dimethylbenz(a) anthracene and, therefore no conclusion could be drawn from experiments performed in the presence of exogenous metabolic activation. No clear indication of gene mutation was seen when CHO cells were treated with test chemical at concentration of 0, 0.1, 0.25, 0.5 or 1.0 mM for 3 hours in the absence of S9 mix. One very diffuse colony was seen in one well out of four at the concentration 0.5 mM, but this colony was a small cluster of apoptotic cells as it was only mildly coloured by crystal violet and, consequently it was considered irrelevant to the present test. This was further supported by the results of the higher tested concentrations of test chemical, i.e. these concentrations did not show any evidence of diffuse or clear colonies present. In addition, no cytotoxic effects were observed when CHO cells were exposed to the test substance at any concentration used. In conclusion, the given read across analogue 2 -methoxynapthalene (CAS no. 93 -04 -9) does not give rise to gene mutations when CHO cells are exposed to the test chemical at 0, 0.1, 0.25, 0.5 or 1.0 mM for 3 hours in the presence and absence of exogenous metabolic activation (-S9 mix). In addition, it does not induce cytotoxic effects at concentrations ≤ 10.0 mM. Hence, it is not mutagenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Ames test:

1. in vitro Gene mutation toxicity study was performed to determine the mutagenic nature of the read across substance 2 -Picoline (CAS no.: 109 -06 -8, E.C. no.: 203 -643 -7). The study was performed using Salmonella typhimurium strains TA97, TA98, TA100, TA102, TA1535 and/or TA1537 in the presence and absence of metabolic activation system. The chemical was used at dose levels upto 5000 µg/plate. Positive and negative controls were tested concurrently. The test chemical did not induce gene mutation in Salmonella typhimurium strains in the presence and absence of metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

2. in vitro Gene mutation toxicity study was performed to determine the mutagenic nature of the read across substance 3 -Picoline (CAS no.: 108 -99 -6, E.C. no.: 203 -636 -9). The study was performed using Salmonella typhimurium strains TA97, TA98, TA100, TA102, TA1535 and/or TA1537 in the presence and absence of metabolic activation system. The chemical was used at dose levels upto 5000 µg/plate. Positive and negative controls were tested concurrently. The test chemical did not induce gene mutation in Salmonella typhimurium strains in the presence and absence of metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

Chromosomal aberration assay:

1. In vitro chromosomal aberration study was performed to determine the mutagenic nature of the read across substance5-Ethyl-2-picoline(CAS no.: 104 -90 -5, E.C. no.: 203 -250 -0). The study was performed using human lymphocytes in the absence of metabolic activation system. The chemical was used at doses 100, 200, 300 and 400 µg/ml. The test chemical did not induce gene mutation in human lymphocyte cells in the absence of metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

2. The cytogenetic effects of the given test chemical1-methyl-3-phenoxybenzene (CAS no.: 3586 -14 -9, E.C. no.: 222 -716 -4)on cultured cells were examined for chromosomal aberration using Chinese hamster cultured cells (CHL / IU) at different concentrations given as: -S9 mix: continuous processing method : 24 h - 0, 50, 100, 200, 400 μg / ml; -S9 mix: continuous processing method : 48 h - 0, 25, 50, 100, 200 μg / ml; -S9 mix: short-time processing method: 0, 50, 100, 200, 400 μg / ml; +S9 mix: short-time processing method: 0, 25, 50, 100, 200 μg / ml. The test substance was dissolved in dimethyl sulfoxide DMSO a stock solution, and then the stock solution was serially diluted with the solvent to prepare a test substance preparation solution of a predetermined concentration. The test substance preparation was added to 0.5 (v / v)% of the culture solution in all tests. As a positive control, the continuous treatment method is 0.03 μg / ml of mitomycin C and the short-time treatment method is benzo [a] pyrene was set to 20 μg / ml. Two hours before the end of culture, colcemid was added to the culture solution to a final concentration of about 0.1 μg / ml. The preparation of chromosome specimens was performed according to the usual method. Two slide specimens were prepared for each dish. The prepared specimens were stained with 3% Giemsa solution for 20 minutes. In order to determine the treatment concentration of the test substance used in the chromosomal aberration test, the effect of the test substance on cell growth was examined. The growth inhibitory effect of the test substance on CHL cells was obtained by counting the number of surviving cells in each group using a hemocytometer, and using the ratio of cell growth to the negative control group as an index. As a result, the concentration showing about 50% growth inhibition of chemical was calculated from the value of 2 concentration with 50%, and it was 176 and 115 μg / ml for 24 hours and 48 hours of continuous treatment respectively. In addition, the concentrations showing about 50% growth inhibition in the presence and absence of S9 mix in the short treatment method were 147 and 234 μg / ml, respectively. According to the results of the cell growth inhibition test, the highest concentration of the test substance used in the chromosomal aberration test is 400 μg / ml in the absence of S9 mix in the 24-hour treatment and the short-time treatment in the continuous treatment method and 48 hours treatment in the continuous treatment In the presence of S9 mix in the short-time treatment method, the concentration was 200 μg / ml. From the above results, it was concluded that the given test chemical did not induce chromosome aberrations in cultured Chinese hamster cultured cells (CHL / IU) in the presence or absence S9 mix.

Mammalian cell gene mutation assay:

1. An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of the given read across analogue 2 -methoxynapthalene (CAS no. 93 -04 -9) when administered to Chinese Hamster Ovary (CHO) cells. The test substance was administered to CHO cells for 3 hours at the dose levels of 0, 0.1, 0.25, 0.5 or 1.0 mM in the absence or presence of exogenous metabolic activation (S9 mix). CHO cells representing the negative controls were exposed to the vehicle. Positive controls, such as N-ethyl-N-nitrosourea (ENU) experiments without metabolic activation and 7,12-dimethylbenz(a) anthracene in experiments with metabolic activation, were also included in each test. The positive control ENU gave a clear indication of gene mutations occurring while no other treatment gave rise to gene toxicity. No mutant colonies were detected following the exposure to positive control of7,12-dimethylbenz(a) anthracene and, therefore no conclusion could be drawn from experiments performed in the presence of exogenous metabolic activation. No clear indication of gene mutation was seen when CHO cells were treated with test chemical at concentration of 0, 0.1, 0.25, 0.5 or 1.0 mM for 3 hours in the absence of S9 mix. One very diffuse colony was seen in one well out of four at the concentration 0.5 mM, but this colony was a small cluster of apoptotic cells as it was only mildly coloured by crystal violet and, consequently it was considered irrelevant to the present test. This was further supported by the results of the higher tested concentrations of test chemical, i.e. these concentrations did not show any evidence of diffuse or clear colonies present. In addition, no cytotoxic effects were observed when CHO cells were exposed to the test substance at any concentration used. In conclusion, the given read across analogue 2 -methoxynapthalene (CAS no. 93 -04 -9) does not give rise to gene mutations when CHO cells are exposed to the test chemical at 0, 0.1, 0.25, 0.5 or 1.0 mM for 3 hours in the presence and absence of exogenous metabolic activation (-S9 mix). In addition, it does not induce cytotoxic effects at concentrations ≤ 10.0 mM. Hence, it is not mutagenic.

2. An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of test chemicalP-tert-butylphenol (CAS no.: 98 -54 -4, E.C. no.: 202 -679 -0)when administered toL5178 TK +/- mouse lymphoma cells. The test was performed according to OECD test guideline 476 adopted 21st July 1997. In the genotoxicity test, test chemical was administered toL5178 TK +/- mouse lymphoma cellsfor 3 hrs at the dose levels of 0, 5, 10, 20, 40 and 60 in the absence or presence of exogenous metabolic activation.The concentration range was selected from the results of a preliminary cytotoxicity test 0, 5, 10, 20, 40, 80 µg/ml , since at 80 µg/ml cytoxicity was observed.The study was performed with both positive and negative controls. Ethylmethylsulphonate (EMS) (-S9) and cyclophosphamide (CP) (+S9) were used as the positive control and DMSO was used as negative control with and without metabolic activation for 3 hours.The response in the negative controls were just outside the range of 1-10 x 10-5 mutants per survivor that is normal for the TK +/- locus in the L5178Y cells but this is likely not to affect the integrity of the test. Both the positive control substances produced marked increases in the mutant frequency per survivor indicating that the test system was operating satisfactorily and that the metabolic activation system was active. The test substance did not induce increases in mutant frequency at any dose level, either with or without metabolic activation. Hence, the test substance was considered to be non-mutagenic. The study was conducted according to the GLP.

Justification for classification or non-classification

Based on the data available, the given test chemical doesnot exhibit gene mutation in vitro by Ames assay, chromosome aberration assay and mammalian cell gene mutation assay. Hence, the test chemical is not likely to classify as agene mutant as per the criteria mentioned in CLP regulation.